The discovery of antimicrobial peptides in insects, lower vertebrates and mammals has unveiled a previously unrecognized component of animal host defense. Antimicrobial peptides are gene-encoded antibiotics with activity against many classes of microbes. Defensins are the predominant family of such peptides in mammals. Studies by our group and others have discovered that defensin peptides are expressed by mammalian mucosal epithelial cells, providing them with the capacity to participate in local host defense. Although the intestinal epithelium is a surface in continual contact with luminal contents variably laden with microbes, infection is uncommon. Our underlying hypothesis is that in humans, the epithelial defensins HD5 and HD6 contribute to antimicrobial defense of the enteric mucosa. In this grant proposal, we will test biological functions of epithelial antimicrobial peptides in vivo through transgenic expression of HD5 and HD6 peptides in mice. We propose that transgenic expression of these human defensins may provide mice with an enhanced capacity to resist bacterial challenges. Based on preliminary studies of our established transgenic mice, the experiments described here will establish a clearer understanding of the contributions of human antimicrobial peptides to innate host defense. To test our hypotheses, Aim 1 will assay the immunological consequences of human HD5 expression in transgenic mice. We will characterize the ability of HD5 transgenic mice, compared to control wild-type mice, to resist enteric infection by Salmonella typhimurium (Aim 1A), and parallel experiments will extend to other enteric pathogens (Aim 1B). We will examine the impact of transgenic HD5 expression on resident microflora of the mouse intestine (Aim 1C). The antimicrobial activity contributed by transgenic expression will be quantitated in vitro, including analysis of isolated crypts (Aim 1D). The HD6 gene and peptide share little sequence identity to HD5, yet they are expressed together in Paneth cells. In Aim 2, we will generate HD6 transgenic mice (Aim 2A) use recombinant HD6 to develop an antibody for immunoassays (Aim 2B), and characterize the transgenic expression of HD6 at the gene and protein level (Aim 2C). We will then characterize the effects of transgenic HD6 expression on resistance to enteric bacterial colonization and infection (Aim 2D) using the approaches developed in Aim 1. Finally, through lineage interbreeding we will create HD5/HD6 compound transgenic mice to determine if these two peptides have synergistic activities in vivo (Aim 2E). The proposed investigations, and other studies of innate immunity, may provide insights yielding novel therapeutic targets and approaches to combat infectious disease.